production and optimization of amylase and glucoamylase ... · ujmr, volume 2 number 1 june, 2017...

UJMR, Volume 2 Number 1 June, 2017

UMYU Journal of Microbiology Research

Production and Optimization of Amylase and Glucoamylase from Aspergillus Niger under Solid State Fermentation for Effective Production of Glucose Syrup

Abalaka, M. Ea* and Adetunji, C. Ob

aDepartment of Microbiology, Federal University of Technology, Minna, Niger State, Nigeria. b Microbiology, Biotechnology and Nanotechnology Laboratory, Department of Biological Sciences,

Landmark University, Omu Aran, Kwara State, Nigeria *Correspondence Author: Abalaka, M. E.

Corresponding authors Email: [email protected]

INTRODUCTION Microbial-derived enzymes are of greater importance in the production of useful products to mankind and they are produced by different types of micro-organisms including bacteria, fungi, and yeast. Production of enzyme from microbial origin are widely accepted because they are Generally Regarded as Safe (GRAS), easily manipulated so as to obtain enzymes with desired characteristics including predictability, controllable and reliable enzyme contents, and their bulk production which is economically feasible when compare with other sources (Sindhu et al 2009). Production of biotechnologically important enzymes involves screening and identification of filamentous fungi capable of secreting extracellular enzymes with greater activities. (Zambare, 2010). Glucoamylase is produced from different type of fungi but the exclusive production of this enzyme in industry have been obtained mainly by Aspergillus niger (Wang et al., 2006), Aspergillus oryzae (Biesebeke et al., 2005), Aspergillus awamori and Aspergillus terreus (Berka et al., 1992) due to their ubiquitous nature and non-fastidious nutritional requirements. Enzymes are majorly produced using solid-state fermentation techniques from microorganisms (Sodhi et al 2005).

The wide usage of the enzymes in the industry include high fructose syrups production, cellulose, leather, detergents, liquor, bread, children cereals, ethanol production, as well as in the pharmaceutical and chemical industries such as the synthesis of optically pure drugs and agrochemicals (Pandey and Radhakrishnan, (1993), Gupta et al. (2003), Norouzian et al. (2006) and Zambare, (2010). Starch syrups are sweet edible products that are widely used in confectionery and other food products. In the United States, these syrups are known as ‘corn syrups' as they are produced by acidic or enzymatic hydrolysis of corn starch. However, in Europe, usage of different starches as starting material, with more accurate description ‘glucose syrups' is preferred because they contain glucose polymers in addition to glucose (Ipek, 2006). Federal Office of Statistics Annual Report (2000) indicated millions of dollar was spent on the importation of sugar syrups into Nigeria. Glucose syrups are essentially industrial sugars used in the manufacture of food products, and are mainly consumed in the confectionery industry (Akinola, 2004). Glucose syrup can be produced from different starch crops such as potato, cassava, rice, maize.

Abstract The continual demand for sugars and coupled with the chemical hazards associated with chemically produced syrup had led to the development of enzymatic technology which has increased the production of glucose syrups most especially using enzymes from microbial origin. The aim of this work was to isolate, screen and optimized the best cultural condition that can lead to the production of glucose syrups from Nigeria cassava starch using enzymes produced by soil microorganisms. Also to determine proximate and physiochemical properties of the glucose syrup produced from yellow and white maize using microbial enzymes. The results obtained from the preliminary screening showed that most of the isolates obtained were Aspergillus niger. It was observed that Aspergillus niger had the largest zone of amylase activity of 35.0 mm when compared to Fusarium pallidorosium that had the lowest (5.0 mm). The optimal conditions for enzyme production; temperature, incubation period, inoculum concentration, and pH were 30 oC, 5 days, 1 × 106 CFU/ml, and pH of 5 respectively for the production of glucoamylase and amylase. Generally, strain AMO1 was observed to produced more glucoamylase when compare to amylase production. The crude protein, the colour, viscosity, ash content, reducing sugar content, pH and carbohydrate contents of the syrup from yellow maize were more than that of the syrup from white maize. Keywords: Glucoamylase; Amylase; Glucose syrups; Cassava; Optimization, Aspergillus niger

Received: 2nd Feb, 2017 Accepted: 9th Apr, 2017

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Corn starch is the major industrial raw material for glucose and fructose syrup production in the US and in many other parts of the world (Cabello, 1999). Cassava (Manihot esculenta), also called manioc or tapioca, is a perennial plant widely grown in many tropical countries, including Nigeria and Brazil on a very large scale. Since cassava roots contain high starch content and low quantity of impurities such as protein and lipid, they are acknowledged as an excellent source of purely suitable starch for a wide range of applications (Tonukari, 2004). Many factories utilize the acid conversion to obtain glucose syrup (especially for the sugar confectionery industry) (Monica and Vionel, 2006). However, the use of enzymes is preferred to acid, because it produces high yields of desired products and less formation of undesired products such as toxic compounds (Silva et al., 2010). MATERIALS AND METHODS Cassava (Manihot utilissima), white and yellow maize were bought from central market Minna, Niger State, Nigeria. All other chemicals used were obtained commercially and are of analytical grade. Extraction of Cassava Starch Cassava was prepared into starch in the laboratory according to standard procedures as described by White et al., (1990) modified by Krieger et al., (1997). Corn kernels were hand-picked and cleaned to remove foreign material, mold, and broken kernels before analysis. Kernels of whole corn (2, 5, or 10 kernels) were steeped in 5 mL of 1% sodium metabisulfite solution (0.67% SO2) at 45°C for 24, 48, or 72 hr, followed by manual removal of the pericarp and germ with forceps. The separated endosperm was placed in a 50-mL centrifuge tube with 10 mL of distilled water and homogenized using a vortex type tissue homogenizer at 20,500 rpm for 30 sec. The homogenized slurry was filtered by using a 30-µm nylon filter under vacuum with several washes, for a total wash water volume of 500 mL. Coarse and fine fibers and part of the protein were removed during filtration. The starch-protein mixture from the filtrate was further separated by either centrifugation or sedimentation. Each sample was separated three times, with 250 mL of distilled water used for each of the three separations. All treatments were performed in replicates of five, and the results averaged. Isolation and Characterization of Fungal Organisms One gram of soil gotten from the boy’s hostel premises of Federal University of Technology

Minna (FUTMINNA), Niger State was mixed in 9ml of sterile distilled water. Aliquot of 1ml of the mixture was transferred to a tube containing 9ml of sterile distilled water. The procedure was repeated to obtain serial dilution and 0.1 ml from the fourth tube (with the dilution of 4-4) was poured into a petri dish then potatoes dextrose agar was poured into the same Petri dish and gently rocked to mix. The plates were incubated at ambient temperature for 48 hours for the fungal growth. Individual colonies were isolated and subcultured on same medium to obtain pure cultures. The fungal isolates were identified according to the taxonomic key of Alexopoulos et al. (1990) on the basis of their macroscopic and microscopic characteristics viewed through a microscope with a magnification of 40 objectives. The organisms were maintained on PDA slants and stored at 4oC. The biochemical and physiological properties of the isolates were confirmed using the protocol of Hewa et al., (2014). Screening of Isolates for Starch Hydrolysis The isolates were screened for starch hydrolyzing ability. The fungal isolates were inoculated on 1 % starch PDA plate. The sterilized medium was cooled and aseptically 300 mg of Norfloxacin per 100 ml of medium was added as antibacterial. Each medium plate was point inoculated with the spores of the fungal isolates and incubated at ambient temperature for five days. The growth pattern and starch hydrolysis were evaluated qualitatively only by visual examination after flooding the plates with iodine solution and measuring the clear zones of starch hydrolysis around the growths of the fungal isolates. Hyper-producer of amylase was chosen and employed in subsequent experiments (Akpan et al., (1999), Alfred, (2007). Inoculum Preparation The best-selected strain was subcultured at 30 ± 2ºC under the dark condition and one week-old-PDA slant culture was used for development of inoculum. A spore inoculum was prepared by adding 10-15 ml of sterile Tween 80 (0.8%) to the slant and shaking vigorously using potato dextrose broth. One ml of spore suspension (1x107spores/ml) was used per flask to carry out solid state fermentation. Production of Amylase and Glucoamylase Enzyme from Maize Malt White and yellow maize samples were washed properly with water and soaked in water for 48 hours. The soaked maize was rinsed and the water changed twice a day to prevent it from fermenting and producing odour.

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The maize was spread on a perforated tray to allow excess moisture drain and give aeration for germination/sprouting for 5 days with intermittent turning and wetting. The sprouted maize was dried in an oven at 50 °C for 48 hours. The dried maize malt was ground into powder and used as a source of amylase enzyme. Production of Amylase and Glucoamylase Enzyme through Solid state fermentation The following procedures were carried out for the production of this enzyme through solid state fermentation using maize as a substrate and inoculated with the isolate that showed the best amylolytic activity in the preliminary screening for hydrolysis of starch. Preparation and Inoculation of Media for Solid State Fermentation Maize was reduced to smaller sizes by pounding and used as a substrate for growth of the selected isolate. Sixty grams of ground maize was weighed and mixed with 45ml of basal salt solution consisting of; (NH4)2SO4, 4g/l, KH2PO4, 2g/l, MgSO4, 1g/l, FeSO4.7H2O, 6.3µg/l, MnSO4, 0.1µg/l, ZnSO4, 6.2µg/l (Bertolin, et al., 2003). The medium was sterilized by autoclaving at 121°C for 15 mins. The pH was adjusted to 4.5 using acid. About 1x107 spores/ml of the inoculum was used to inoculate the media contained in a conical flask (Patel, et al., 2009). The culture was incubated at 30°C for fermentation to take effect for 7 days. Extraction of Glucoamylase and Amylase Enzyme Produced Through Solid State Fermentation After 7 days of fermentation, the whole mixture was treated with 100ml of cold (4°C) distilled water. It was stirred and agitated thoroughly for 30 min then filtered through muslin cloth. The residue was again treated with 100ml distilled water in the same way and filtered. The filtrate was centrifuged at 6000 rpm for 20minutes. The supernatant obtained was again filtered through Whatman filter paper No. 1 so as to obtain a cell-free supernatant which was used as a source of crude enzyme for assaying glucoamylase and amylase(Mahmoud et al., 2007). Enzyme assay Two grams of yellow and white maize malt powder used as a source of crude enzyme was dissolved in 20ml of citrate buffer each. The mixture was stirred and agitated for 10mins then filtered. Two millilitres of each filtrate was pipetted into a test tube. Aliquot of 1ml of 1% starch solution was added into the test tube and incubated at 40°C for 30mins. Alpha-amylase and glucoamylase were obtained by incubating 1% of soluble starch and 1% maltose respectively in 0.1 M citrate buffer of pH 5.0 at

45°C for 30 min. Two milliliters of DNS solution was added to each of the test tubes and the tubes were immersed in boiling water bath (100°C) for 5mins to stop the reaction. The tubes were cooled. The final volume was brought to 20ml with distilled water. The absorbance for all the test tubes was measured with a spectrophotometer at 540nm and compared with a standard curve prepared using 0.10 to 1.0 mg of glucose/ml. Enzyme activities were expressed in International Units (IU). One IU is defined as one μmol of glucose (for amylase and glucoamylase activity) equivalents released per minute per ml under the standard assay conditions by using glucose standard curve (Silva et al 2005). Appropriate dilution factors were used during the estimation of enzyme activity. Absorbance was measured at 540 nm using the UV-Visible spectrophotometer. Alpha-amylase and glucoamylase activity were determined according to the method reported by Tang-um et al., (2012). Hydrolysis of Gelatinized Starch using Amylase and glucoamylase This was carried out using modified method of Nweke and Abiamere, (2014), 100g of cassava starch was weighed accurately and transferred into a clean 1000ml beaker. One hundred milliliters of distilled water was measured with a measuring cylinder and poured into the beaker containing the measured cassava starch. The mixture was stirred thoroughly to produce a white slurry paste of cassava starch and water. Five hundred milliliters of boiling distilled water was added to the starch slurry in the beaker while stirring simultaneously. The slurry was continuously stirred for 10 mins until all the starch was gelatinized forming a thick, translucent paste. Twenty grams of maize malt powder used as a source of amylase was measured and poured into the gelatinized starch paste. The mixture was stirred continuously until liquefaction starts to take place. The mixture was immersed in a water bath to maintain the temperature at 90 °C in order for complete liquefaction to take place. This was allowed for 1hr with intermittent stirring.After liquefaction, the mixture was allowed to cool. The pH was adjusted to 4.5 with acid while monitoring with a pH meter. The temperature was confirmed to be 60 °C with the aid of a thermometer. Twenty milliliters of enzyme filtrate obtained through solid state fermentation by Aspergillus niger was introduced into the mixture as the Glucoamylase and amylase sources.

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The mixture was stirred and maintained at 60°C for 3hrs in order for saccharification to take place so as to allow the breakdown the entire remaining starch, maltose, and other dextrins into glucose. Thereafter a cheesecloth was used to extract the syrup-like filtrate. The beakers containing syrup-like filtrate were then transferred to a fume cupboard for evaporation and concentration. The pH of the resulting concentrated glucose syrup was determined. The Brix was measured with a refractometer, and the glucose content was determined using Fehling's method of glucose estimation. Proximate Analysis and physicochemical properties of Glucose Syrup This was carried out according to the method of A.O.A.C (1990). The proximate analysis and physicochemical properties of Glucose Syrup such as water activity, total soluble solids and pH, colour, moisture, protein, crude fiber, fat, ash and carbohydrate contents were determined. Effect of incubation temperature on enzyme production The effect of incubation temperature on enzyme production was determined by incubating the inoculated (with 1x107 density) flasks having 5 g of the substrate from yellow or white maize with 20 ml of mineral medium of pH 5 at different temperatures ranging from 15, 25, 30, 40, 50 and 55 °C for 5 days in a biological oxygen demand (BOD) incubator. Effect of inoculum concentration on enzyme production The effect of inoculum size based on the number of spores was studied using the spore concentration of 1x104, 1x105, 1x106, 1x107 and 1x1081x109, 1x1010spore ml-1 of Tween-80 (Kheng and Omar 2005). The flasks having the sterile basal medium were inoculated with the different spore suspensions and were incubated at 30°C on a BOD shaker cum incubator for 96 h and then the partially purified enzymes produced were used for the evaluation of the enzyme activities. Effect of incubation period on enzyme production Flasks containing 5 g of substrate from yellow or white maize were inoculated with 1 ml (107 spores/ ml) of spore suspension in 20ml of basal medium and incubated at ambient temperature. The enzymes were obtained and assayed after 3, 4, 5, 6,7days interval. Effect of initial pH on enzyme production The effect of initial pH on enzymes production was investigated by adjusting the initial pH of Mineral medium to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0. The flasks containing 5 g of substrate

from yellow or white maize and 20 ml of sterile mineral medium were inoculated with 1 ml of spore suspension (107 spores) and incubated for 5 days at 28 ± 2 °C in a BOD incubator and enzyme activities determined. RESULTS Isolation and Preliminary Screening of Isolates The soil obtained from FUTMINNA showed the various diversities of fungi present in the ecological niche with many isolates that can be utilized for amylase and glucoamylase production. They were fully characterized using cultural and morphological characteristics (Table 2). The isolate was further screen to select the best isolate that can hydrolyze starch with the aid of their amylolytic enzyme. It was observed that Aspergillus niger had the largest zone of clearing of 35.0 mm when compared to Fusarium pallidorosium that had the lowest clear zone of 5.0 mm (Table 4). The best-screened isolate with the most amylolytic activity on the starch agar was then coded Aspergillus niger AM01 (Fig 1). Microscopic examination was later used to confirm the features of the most active strain. It was observed that it had a conidial head, radiate, tending to split into loose columns with age. Conidiophore stripes had a smooth-walled, hyaline but often in brown colour (Plate 2). Biochemical and physiological characterization of isolates The biochemical and physiological test carried out revealed that Aspergillus niger AM01 could utilize dextrose, dextrose, fructose, lactose, maltose, mannitol, sucrose, starch, xylose as a carbon source but it was observed that sorbitol, adonitol, inositol, raffinose were not utilized by the organism. The best incubation evaluation carried out showed that Aspergillus niger AM01 can grow from 20oC to 35oC while the optimum temperature was obtained at 30oC. It can also grow at pH ranges of 5 to 10. Moreover, Aspergillus niger AM01 can grow at the salt concentration ranging from 1 % to 4 % (Table 3). Optimization of Amylase and Glucoamylase Production The best optimum condition was determined when different substrate was utilized (yellow and white maize). It was observed that the best temperature of 30oC produced the optimum glucoamylase and amylase enzyme of 7.6 IU and 4.3 IU when yellow maize was used as substrate but glucoamylase and amylase enzyme of 5.1 IU and 2.3 IU was obtained when white maize was used.

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Also, the best incubation period was on 5 day with optimum glucoamylase and amylase enzyme of 7.5 IU and 4.2 IU obtained when yellow maize was used as substrate but glucoamylase and amylase enzyme of 4.4 IU and 2.3 IU was obtained when white maize was used. The best inoculum concentration that favours the best enzymes activities was 1 × 106 CFU/ml. It was discovered that the optimum glucoamylase and amylase enzyme of 6.3 IU and 4.1 IU were obtained when yellow maize was used as substrate but glucoamylase and amylase enzyme of 3.7 IU and 2.5IU was obtained when white maize was used. Moreover, the optimum pH was pH of 5. It was observed that the optimum glucoamylase and amylase enzyme of 6.5 IU and 4.2 IU were obtained when yellow maize was used as substrate but glucoamylase and amylase

enzyme of 5.1 IU and 3.2 IU was obtained when white maize was used (Table 3 and 4). Production of Glucose Syrup The most active screened strain Aspergillus niger AM01 was then utilized for the conversion of gelatinized cassava flour to glucose syrup using sprouted white and yellow maize Plate 3. Proximate Analysis and Physicochemical Properties of Glucose Syrup The Crude protein and Carbohydrate values of the syrup from yellow maize enzymes was more than the syrup from white maize (Table 4). 0.50±0.2b, 0.75±0.4a crude protein for white maize amylase and glucoamylase values and 0.50±0.1b, 0.76±0.3a values for yellow maize respectively. The carbohydrate values for both maize types for amylase and glucoamylase are 13.10±3.1c, 13.25±2.5c and 14.25±3.3b and 15.20±2.6a respectively.

Table 2: Preliminary screening of various strains of fungi isolated for amylase production

Means with different superscripts within the same column are significantly (P=0.05) different

Fig1: A: Aspergillus niger AM 01 B. Colour change after treatment growing on Starch agar with lugol’s iodine

S/N Fungus Amylase Activity (Clear zone mm)

1 Rhizopus stolonifer 10.0±1.1b 2 Saccharomyces cerevisiae 3.0±0.5d 3 Aspergillus niger 35.0±2.8a 4 Aspergillus flavus 12.0±1.0b 5 Fusarium oxysporium 8.0±0.5bc 6 Fusarium pallidorosium 5.0±0.3cd

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Table 2: Characterization of the isolated fungi.

Isolate code No

Characteristics Probable fungus

1 Colony whitish becoming grayish-brownish sporangiophores and brown-black sporangia, often over 20mm high. Sporangiophores are colourless to dark brown, smooth or slightly rough-walled stolons opposite the branched rhizoids. Sporangia globose to subglobose, ovoid, blackish-brown at maturity.

Rhizopus stolonifer

2 Colonies of Saccharomyces grow rapidly and mature in three days. They are flat, smooth, moist, glistening or dull, and cream to tannish cream in color. Blastoconidia (cell buds) are observed. They are unicellular, globose, and ellipsoid to elongate in shape. Multilateral (multipolar) budding is typical.

Saccharomyces cerevisiae

3 Colonies at 25oC attaining a diameter of 4-5cm within 7 days, usually consisting of a compact white or yellow basal felt with a dense layer of dark brown to black conidiophres. Conidial heads, radiate, tending to split into loose columns with age. Conidiophore stipes smooth-walled, hyaline but often in brown colours. Vesicles globose to subglobose.

Aspergillus niger

4 Colonies at 25 oC attaining a diameter of 3-5cm within 7 days, usually consisting of a dense felt of yellow-green conidiophores. Conidia heads, radiate tending to split into loose columns with age. Conidiophores stipes smooth-walled, hyaline but often in brown colours.

Aspergillus flavus

5 Colony at 25oC attaining a diam. of 4.5 cm in 4 days. Aerial mycelium sparse or floccose, becoming felting, whitish or peach, usually with the purple tinge more intense near the medium surface. Variable in shape and size, ovoid-ellipsoidal to cylindrical, straight or slightly curved.Conidiophores are usually short branched on phialides.

Fusarium exosporium

6 Colonies 25 oC attaining a diameter of 6cm in 4 days, white often with a peach tinge. Aerial mycelium floccose, whitish or peach often changes to brownish. Sporodochia absent. Conidia 3-5(7) septate fusiform straight or somewhat curved, wedged shaped, apical cell beaked, chlamydospore often sparse, hyaline, smooth-walled globose.

Fusarium pallidorosium

Table 3: Biochemical and physiological characteristics of Aspergillus niger AM 01

+++ = Utilized strongly, ++ = Utilized moderately, + = Utilized slightly,- = No activity

Sugars Acid production from sugars

temperature (oC)

Growth at different temperatures

pH Growth at different pH

NaCl (%) Growth at different NaCl conc.

Sorbitol - 5 - 4 - 1 +

Adonitol - 10 - 5 + 2 +++

Dextrose + 20 + 6 + 3 ++

Fructose + 25 ++ 7 + 4 ++

Inositol - 30 +++ 8 + 5 -

Lactose + 35 ++ 9 + 6 -

Maltose + 40 - 10 + 7 -

Mannitol + 45 - 8

Raffinose - 50 - 9 -

Sucrose + 10 -

Xylose +

Starch +

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Plate 2: Photomicrograph of Aspergillus niger AM01. X 40 A: Mature sporangium with hyphae, B: Conidial heads C: Spores D: Hypae E: Conidophore stripes with smooth-walled

A B C

D E

A B C

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Plate 3: Production of Glucose syrup from Cassava flour A: Sprouted white maize B: Sprouted yellow maize C: Solid state fermentation D:intermittent stirring E: Complete liquefaction F: Syrup from white maize G: Syrup from yellow maize; H: Process of pH adjustment.

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Fig 1: Influence of different factors on enzyme activities when yellow maize was used as a substrate; A = pH, B=Incubation days, C=Inoculum development, D = Temperature. Summary: Glucoamylase: 1x106, day 5, pH=5, temperature= 30 oC; Amylase: 1x106, day 5, pH=5, temperature= 30 oC

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Fig 2: Influence of different factors on enzyme activities when White maize was used as a substrate; A=Inoculum development, B=Incubation days, C = pH, D = Temperature. Summary: Glucoamylase: 1x106, day 5, pH=5, temperature= 30 oC; Amylase: 1x106, day 5, pH=5, temperature= 30 oC Table 4: Proximate, Physicochemical Properties and Enzyme Activities of Syrup Produced by AspergillusnigerAM01 using yellow and white maize

Proximate and Physicochemical paprameters

Amylase activity (mm) Glucoamylase activity (mm)

White maize

Yellow maize

Control White maize Yellow maize

Control

Moisture 84.0±3.2bc 82.0±4.1c 98.0±2.9a 86.0±3.2bc 88.0±2.6b 95.0±3.1a Ash 0.23±0.1c 0.22±0.1c 0.85±0.3a 0.25±0.1c 0.26±0.1c 0.75±0.2b Protein 0.50±0.2b 0.50±0.1b 0.41±0.1bc 0.75±0.4a 0.76±0.3a 0.31±0.1c Carbohydrate 13.10±3.1c 13.25±2.5c 6.4±1.1e 14.25±3.3b 15.20±2.6a 8.4±0.6d Fat 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 Fiber 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 0.00±0.0 Sulphate 1.36±0.2bc 1.28±0.9c 1.62±0.3a 1.42±0.2b 1.45±0.1b 1.60±0.3a Viscosity (%) 56.4±3.5d 62.1±3.2c 38.6±2.8f 69.2±3.7b 73.4±5.1a 43.8±3.9e pH 5.68±0.4a 5.53±0.3a 4.2±0.4b 6.0±0.2a 6.3±0.2a 5.7±0.3a Brix 8.7±0.6c 9.4±0.5b 6.8±0.4d 10.9±0.8a 11.3±0.4a 6.3±0.2e Reducing sugar(mg/ml)

20.0±1.0bc 22.0±0.9ab 24.0±2.1a 16.0±2.0d 18.0±1.3cd 22.0±0.8ab

Enzyme activities

5.0±0.2b 5.4±0.1b 1.4±0.2d 7.5±0.8a 7.9±0.4a 2.8±0.6c

Means with different superscripts within the same row are significantly (P=0.05) different DISCUSSION The strain used during this study showed that it has the ability to utilized starch and other sugars as a source of carbon and energy. The result obtained during this study by Mchunu et al., (2013) observed that monosaccharides showed good assimilation by Trichoderma reesei and Aspergillus niger. Aspergillus niger AM 01 was observed to possess amylopectin enzyme which enhances it starch degrading

ability. These findings are in line with the research conducted by the various researcher (Omemu et al., 2005; Pandey et al., 2006 and Sasi et al., 2010) where the collection of potent species was made by plate method. However, zones of clearing was associated with the amount of enzyme formed (amylase) during analysis of enzyme activity using starch as a sole source of carbohydrate.

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The amylolictic property of Aspergillus niger AM 01 isolated from soil of FUTMINNA was check by blue-black coloration obtained after flooding the plate with starch iodine. The occurrence of amylolytic organisms from the soil agrees with earlier reports of Adebiyi and Akinyanju (1998) that the soil is known to be a repository of amylase producers. Aspergillus niger AM 01was selected among all the isolated strains on starch agar. This is because it gave a direct visual indication of starch hydrolysis (Akpan et al., 1999a). This study showed that the utilization of raw starch to by the crude enzymes of A. niger AM 01 is significantly dependent on the starch source and time of incubation. This agrees with earlier reports of Okolo et al., (1995) that the susceptibility of starch granules to digestion by amylase is dependent on starch source and length of amylase or glucoamylase treatment. The high digestibility of raw cassava starch observed in this study is similar to the findings of Okolo et al., (1995). The ability of the crude amylase of A. niger AM01 to hydrolyze the root starches especially cassava starch presents a remarkable property since these root starches are abundantly available in the tropics. According to Anthony et al., (1996) and Oluwole et al., (1999), over 30 million tons of cassava is lost yearly since cassava is perishable after harvesting. Conversion of raw cassava by

this enzyme means that some of the cassava could be used as raw materials by the starch industry for value-added products. This will reduce wastage and improve economic gains. The result obtained from the proximate analysis is in line with Oino Food Ltd. (2004). CONCLUSION The fungal enzyme from Aspergillus niger using white maize as the substrate for solid state fermentation has proven to be a good tool for the enzyme hydrolysis of cassava starch for the production of glucose syrup. This can be harnessed by industries for the commercial production of glucose syrup. It was concluded from this study that the enzymes, glucoamylase, and amylase, produced by A. niger both isolated from FUTMINNA soil showed optimal characteristics, such as optimum pH and temperature and as well good thermal stability, that can be used in the industrial process such as starch processing. The amylolytic enzymes produced by A. niger AM01 are suitable for biotechnological applications, considering the high productivity of these enzymes, the good capacity to degrade starch and its derivates. Furthermore, the technology used for glucose and fructose syrup production using Nigeria cassava starch and enzymes produced by soil microorganisms was successful and can be carried out on an industrial scale.

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